This invention relates to preparations which are useful in inhibiting the proliferation of human tumor cells and more particularly is directed to macrophage derived factors which cause release of intracellular iron and thereby inhibit catalytic activity of enzymes with iron-sulfur centers.
Cancer is a very wide spread and severe health problem which affects millions of people yearly, resulting in debilitating symptoms and often death. Numerous approaches, often fruitless, have been taken by medical scientists in an attempt to identify substances which may be of some usefulness in slowing or stopping the growth of human tumors. One avenue which has shown some promise is through the stimulation of the afflicted patient's immune system, thereby inducing the patient's immune system to produce substances capable of reducing the growth rate of tumor cells or, hopefully, killing them outright. Unfortunately, such an approach is often of little use in that the immune systems of cancer patients are either over burdened already or are simply incapable of responding to such immuno-stimulation.
This has led researchers to attempt to identify and purify substances produced by the immune systems of immuno-competent animals which are active in slowing or stopping the growth rate of tumor cells. Included in such studies have been attempts to identify substances produced by "activated" macrophages, that is, macrophages which have been stimulated by some immuno-stimulating agent to produce tumor-suppressing substances. Indeed, a number of tumor-suppressive substances have been identified from activated macrophages.
Furthermore, numerous in vitro studies demonstrate that coculture of activated macrophages with tumor cells leads to cytolysis or cytostatis of the tumor cells. Although the cytolytic response has been more extensively studied, macrophage induced cytostasis is more frequently observed and is believed to play an important role in controlling the development, progression and spread of tumor cells.
One cytostatic mechanism of activated macrophages is mediated through lesions induced by the effector cell in the target cell mitochondrial electron transport chain (ETC), in particular at Complex I and II. Such lesions result in growth inhibition or, if the damaged cells are not able to conduct adequate levels of glycolysis, death of the target. Kilbourn and co-workers, (1984) J. Immunol., 133:2577, have demonstrated that this cytotoxic mechanism might be accounted for by the secretion of a monokine, termed respiration inhibitory factor (RIF), which appears in large measure to mimic the activated macrophage in the exertion of cytostatic effects on a number of tumor cells.
Studies demonstrating release of intracellular iron by tumor cells provide evidence for a second pathway of macrophage-mediated cytostasis. These studies show a temporal correlation between iron release and inhibition of DNA synthesis in tumor cells cocultured with cytotoxic activated macrophages. Recently, studies by Hibbs et al., (1986), J. Clin. Invest., 78:790 have demonstrated a mechanism whereby macrophage-induced iron release could affect tumor cell metabolism.
Using a macrophage-tumor cell cocultivation system, Hibbs et al. showed that macrophage induced release of intracellular iron from tumor cells was associated with inhibition of the Krebs cycle enzyme aconitase, one of a class of enzymes having an iron-sulfur center essential for catalytic activity. Activity of the enzyme was restored by culturing the injured tumor cell in a medium supplemented with ferrous iron and a reducing agent, an observation suggesting that the inhibition was mediated by removal of an iron atom from the iron-sulfur center of the enzyme. Thus, macrophage-induced iron release has been causally linked to inhibition of at least one enzyme with an iron-sulfur center. Furthermore, studies demonstrating that macrophage mediated iron release continues long after the inhibition of aconitase has been reported to be complete suggest that lesions aside from the one at aconitase may exist.
Despite the potential importance of macrophage mediated iron release as a mechanism for inducing tumor cell cytostasis, previous studies of the phenomenon have employed cytotoxic macrophage coculture systems rather then attempting to identify whether a soluble factor per se was being expressed by the macrophages.
In contrast, the present invention provides for a preparation containing a soluble factor or factors capable of mediating iron release from tumor cell targets without the need for the presence of macrophages. Since this preparation induces release of iron from tumor cells without the need for macrophage coculture , it is likely to prove important as an inhibitor of tumor cell growth.